Project Summary/Abstract Antimicrobial resistance threatens treatment of infectious diseases and medical procedures that depend on prevention of infection. Since the development of new antibiotics is slow and costly, this proposal concentrates on the search for strategies to extend the useful life of currently available drugs. The aminoglycoside 6?-N- acetyltransferase type Ib [AAC(6?)-Ib] is responsible for most cases of resistance to amikacin (Ak) and other aminoglycosides in Gram-negative pathogens. The dissemination of the aac(6?)-Ib gene among these pathogens is eroding the efficacy of these antibiotics that are an important component of the armamentarium against severe infections. The long-term goal of this research is to develop compounds that in combination with Ak reduce resistance to susceptibility levels and can be used as adjuvants to treat Ak-resistant infections. One objective of this project is to design oligonucleotide analogs, known as external guide sequences (EGSs), that bind a complementary region of the aac(6?)-Ib mRNA and form a substrate for RNase P, which cleaves the mRNA preventing translation. Previous experiments identified LNA/DNA chimeric oligonucleotides that induce degradation of the mRNA in vitro. However, they must be attached to a cell penetrating peptide (CPP) to reach the cytoplasm for efficient in vivo activity. The experiments described in Specific Aim 1 are designed to identify the CPPs that most efficiently transport the LNA/DNA chimeric oligonucleotides across the cell envelope without interfering with their inhibitory activity. These compounds will be used in combination with Ak to induce phenotypic conversion to susceptibility to Ak. However, the use of EGS technology may result in a reduction, but not elimination, of AAC(6?)-Ib expression. The experiments described in Specific Aim 2 will identify inhibitors of the acetylating reaction that act synergistically with the mix EGS/Ak. These compounds could enhance the action of the mix EGS/Ak or replace the EGS if they are potent enough. Specific Aim 3 consists of testing the effect of combinations of Ak and the different compounds identified in the previous specific aims on an E. coli laboratory strain as well as on clinical Klebsiella pneumoniae and Acinetobacter baumannii isolates that naturally harbor aac(6?)-Ib. Tests will include inhibition of growth of cells in culture, time-kill assays, and treatment of infections in the Galleria mellonella infection model. The most promising compounds will also be assayed against more aac(6?)-Ib-containing clinical isolates and will be tested to determine their cytotoxicity.